Polypeptides having endoglucanase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/675,601, filed Apr. 27, 2005, which application is incorporatedherein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under NREL SubcontractNo. ZCO-30017-02, Prime Contract DE-AC36-98GO10337 awarded by theDepartment of Energy. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides havingendoglucanase activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods for producing and using the polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiohydrolase Iis a 1,4-D-glucan cellobiohydrolase (E.C. 3.2.1.91) activity whichcatalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellotetriose, or any beta-1,4-linked glucose containing polymer,releasing cellobiose from the reducing ends of the chain.Cellobiohydrolase II is a 1,4-D-glucan cellobiohydrolase (E.C. 3.2.1.91)activity which catalyzes the hydrolysis of 1,4-beta-D-glucosidiclinkages in cellulose, cellotetriose, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the non-reducing ends ofthe chain. Cellobiose is a water-soluble beta-1,4-linked dimer ofglucose. Beta-glucosidases hydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

Kvesitadaze et al., 1995, Applied Biochemistry and Biotechnology 50:137-143, describe the isolation and properties of a thermostableendoglucanase from a thermophilic mutant strain of Thielavia terrestris.Gilbert et al., 1992, Bioresource Technology 39: 147-154, describe thecharacterization of the enzymes present in the cellulose system ofThielavia terrestris 255B. Breuil et al., 1986, Biotechnology Letters 8:673-676, describe production and localization of cellulases andbeta-glucosidases from Thielavia terrestris strains C464 and NRRL 8126.

It would be an advantage in the art to identify new endoglucanaseshaving improved properties, such as improved hydrolysis rates, betterthermal stability, reduced adsorption to lignin, and the ability tohydrolyze non-cellulosic components of biomass, such as hemicellulose,in addition to hydrolyzing cellulose. Endoglucanases with a broad rangeof side activities on hemicellulose can be especially beneficial forimproving the overall hydrolysis yield of complex, hemicellulose-richbiomass substrates.

It is an object of the present invention to provide improvedpolypeptides having endoglucanase activity and polynucleotides encodingthe polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingendoglucanase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence which has at least60% identity with the mature polypeptide coding sequence of SEQ ID NO:2;

(b) a polypeptide which is encoded by a nucleotide sequence whichhybridizes under at least medium stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) a complementary strand of (i) or (ii); and

(c) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having endoglucanase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence which has at least 60% identity with the mature polypeptide ofSEQ ID NO: 2;

(b) a polynucleotide having at least 60% identity with the maturepolypeptide coding sequence of SEQ ID NO: 1; and

(c) a polynucleotide which hybridizes under at least low stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a complementary strand of (i)or (ii).

In a preferred aspect, the mature polypeptide is amino acids 18 to 336of SEQ ID NO: 2. In another preferred aspect, the mature polypeptidecoding sequence is nucleotides 52 to 1008 of SEQ ID NO: 1.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides.

The present invention also relates to methods for producing such apolypeptide having endoglucanase activity comprising: (a) cultivating arecombinant host cell comprising a nucleic acid construct comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of using the polypeptideshaving endoglucanase activity in detergents and in the conversion ofcellulose to glucose.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to a nucleotide sequence encoding a signal peptide comprising orconsisting of amino acids 1 to 17 of SEQ ID NO: 2, wherein the gene isforeign to the nucleotide sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the cDNA sequence and the deduced amino acidsequence of a Thielavia terrestris NRRL 8126 endoglucanase (CEL7F) (SEQID NOs: 1 and 2, respectively).

FIG. 2 shows a restriction map of pTter7F.

FIG. 3 shows a restriction map of pAILo1.

FIG. 4 shows a restriction map of pBANe10.

FIG. 5 shows a restriction map of pAILo2.

FIG. 6 shows a restriction map of pAILo22.

FIG. 7 shows the relative conversion of beta-glucan (1% w/v) after 2hours of hydrolysis at pH 5.5 and 60° C.

FIG. 8 shows the relative conversion of beta-glucan (1% w/v) after 24hours of hydrolysis at pH 5.5 and 60° C.

DEFINITIONS

Endoglucanase activity: The term “endoglucanase activity” is definedherein as an endo-1,4-beta-D-glucan 4-glucanohydrolase (E.C. No.3.2.1.4) which catalyses endohydrolysis of 1,4-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixedbeta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and otherplant material containing cellulosic components. For purposes of thepresent invention, endoglucanase activity is determined usingcarboxymethyl cellulose (CMC) hydrolysis according to the procedure ofGhose, 1987, Pure and Appl. Chem. 59: 257-268. One unit of endoglucanaseactivity is defined as 1.0 μmole of reducing sugars produced per minuteat 50° C., pH 4.8.

In a preferred aspect, the polypeptides of the present invention havingendoglucanase activity further have enzyme activity toward one or moresubstrates selected from the group consisting of xylan, xyloglucan,arabinoxylan, galactan, galactomannan, dextran, and chitin. The activityof the polypeptides having endoglucanase activity on thesepolysaccharide substrates is determined as the relative amount of dyereleased from different AZCL-dyed substrates after incubating thesubstrates (5 g per liter) with a polypeptide having endoglucanaseactivity of the present invention (1 mg protein per g of substrate) for1 and 92 hours without stirring at pH 5.0 (50 mM sodium acetate) and 50°C. The release of dye was determined by measuring the absorbance at 590nm.

In a more preferred aspect, the polypeptides of the present inventionhaving endoglucanase activity further have enzyme activity toward xylan.In another more preferred aspect, the polypeptides of the presentinvention having endoglucanase activity further have enzyme activitytoward xyloglucan. In another more preferred aspect, the polypeptides ofthe present invention having endoglucanase activity further have enzymeactivity toward arabinoxylan. In another more preferred aspect, thepolypeptides of the present invention having endoglucanase activityfurther have enzyme activity toward galactan. In another more preferredaspect, the polypeptides of the present invention having endoglucanaseactivity further have enzyme activity toward galactomannan. In anothermore preferred aspect, the polypeptides of the present invention havingendoglucanase activity further have enzyme activity toward dextran. Inanother more preferred aspect, the polypeptides of the present inventionhaving endoglucanase activity further have enzyme activity towardchitin. In another more preferred aspect, the polypeptides of thepresent invention having endoglucanase activity further have enzymeactivity toward xylan, xyloglucan, arabinoxylan, galactan,galactomannan, dextran, and chitin.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the endoglucanase activity of thepolypeptide consisting of the amino acid sequence shown as amino acids18 to 336 of SEQ ID NO: 2.

Family 7 glycoside hydrolase or Family GH7: The term “Family 7 glycosidehydrolase” or “Family GH7” or “CEL7F” is defined herein as a polypeptidefalling into the glycoside hydrolase Family 7 according to Henrissat B.,1991, A classification of glycosyl hydrolases based on amino-acidsequence similarities, Biochem. J. 280: 309-316, and Henrissat B., andBairoch A., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively associated. It is, therefore,preferred that the substantially pure polypeptide is at least 92% pure,preferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 96% pure, morepreferably at least 97% pure, more preferably at least 98% pure, evenmore preferably at least 99%, most preferably at least 99.5% pure, andeven most preferably 100% pure by weight of the total polypeptidematerial present in the preparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having endoglucanase activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation, etc.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software(DNASTAR, Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10 and gap length penaltyof 10. Pairwise alignment parameters are Ktuple=1, gap penalty=3,windows=5, and diagonals=5.

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of SEQ ID NO: 2 or a homologous sequencethereof, wherein the fragment has endoglucanase activity. Preferably, afragment contains at least 270 amino acid residues, more preferably atleast 285 amino acid residues, and most preferably at least 300 aminoacid residues, e.g., amino acids 18 to 336 of SEQ ID NO: 2.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of SEQ ID NO: 1 or a homologous sequence thereof, wherein thesubsequence encodes a polypeptide fragment having endoglucanaseactivity. Preferably, a subsequence contains at least 810 nucleotides,more preferably at least 855 nucleotides, and most preferably at least900 nucleotides.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide which is at least 20% pure, preferablyat least 40% pure, more preferably at least 60% pure, even morepreferably at least 80% pure, most preferably at least 90% pure, andeven most preferably at least 95% pure, as determined by agaroseelectrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having endoglucanase activity.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG and TGA. The coding sequence may be aDNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 2 or a homologous sequence thereof as well as geneticmanipulation of the DNA encoding that polypeptide. The modification canbe substitutions, deletions and/or insertions of one or more amino acidsas well as replacements of one or more amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having endoglucanase activity produced by anorganism expressing a modified nucleotide sequence of SEQ ID NO: 1 or ahomologous sequence thereof, or the mature coding region thereof. Themodified nucleotide sequence is obtained through human intervention bymodification of the nucleotide sequence disclosed in SEQ ID NO: 1 or ahomologous sequence thereof, or the mature coding region thereof.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having EndoglucanaseActivity

In a first aspect, the present invention relates to isolatedpolypeptides comprising an amino acid sequence which has a degree ofidentity to the mature polypeptide of SEQ ID NO: 2 of at least 60%,preferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 97%, 98%, or 99%, which haveendoglucanase activity (hereinafter “homologous polypeptides”). In apreferred aspect, the homologous polypeptides have an amino acidsequence which differs by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has endoglucanase activity. In a preferred aspect,a polypeptide comprises the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 2. In another preferred aspect, a polypeptide comprisesamino acids 18 to 336 of SEQ ID NO: 2, or an allelic variant thereof; ora fragment thereof that has endoglucanase activity. In another preferredaspect, a polypeptide comprises amino acids 18 to 336 of SEQ ID NO: 2.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragmentthereof that has endoglucanase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, a polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, a polypeptideconsists of amino acids 18 to 336 of SEQ ID NO: 2 or an allelic variantthereof; or a fragment thereof that has endoglucanase activity. Inanother preferred aspect, a polypeptide consists of amino acids 18 to336 of SEQ ID NO: 2.

In a second aspect, the present invention relates to isolatedpolypeptides having endoglucanase activity which are encoded bypolynucleotides which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the genomic DNA sequence comprising the maturepolypeptide coding sequence of SEQ ID NO: 1, (iii) a subsequence of (i)or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequenceof SEQ ID NO: 1 contains at least 100 contiguous nucleotides orpreferably at least 200 contiguous nucleotides. Moreover, thesubsequence may encode a polypeptide fragment which has endoglucanaseactivity. In a preferred aspect, the mature polypeptide coding sequenceis nucleotides 52 to 1008 of SEQ ID NO: 1.

The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having endoglucanase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 14, preferably at least 25,more preferably at least 35, and most preferably at least 70 nucleotidesin length. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Even longer probes may be used, e.g., nucleicacid probes which are at least 600 nucleotides, at least preferably atleast 700 nucleotides, more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having endoglucanaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1 or a subsequence thereof, the carriermaterial is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO: 1, thegenomic DNA sequence comprising SEQ ID NO: 1, its complementary strand,or a subsequence thereof, under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 52 to 1008 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencewhich encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 1. In another preferredaspect, the nucleic acid probe is the polynucleotide sequence containedin plasmid pTter7F which is contained in E. coli NRRL B-30837, whereinthe polynucleotide sequence thereof encodes a polypeptide having lipaseactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTter7F which iscontained in E. coli NRRL B-30837.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of SEQ ID NO: 2 or a homologous sequencethereof; or the mature polypeptide thereof. Preferably, amino acidchanges are of a minor nature, that is conservative amino acidsubstitutions or insertions that do not significantly affect the foldingand/or activity of the protein; small deletions, typically of one toabout 30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up toabout 20-25 residues; or a small extension that facilitates purificationby changing net charge or another function, such as a poly-histidinetract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,endoglucanase activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, such as aminoacids 18 to 336 of SEQ ID NO: 2, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

Sources of Polypeptides Having Endoglucanase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide having endoglucanase activity; or a Streptomyces polypeptidehaving endoglucanase activity, e.g., a Streptomyces lividans orStreptomyces murinus polypeptide having endoglucanase activity; or agram negative bacterial polypeptide, e.g., an E. coli or a Pseudomonassp. polypeptide having endoglucanase activity.

A polypeptide of the present invention may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having endoglucanase activity; or more preferably afilamentous fungal polypeptide such as an Acremonium, Aspergillus,Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces,Thermoascus, Thielavia, Tolypocladium, or Trichoderma polypeptide havingendoglucanase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having endoglucanaseactivity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride polypeptide havingendoglucanase activity.

In another preferred aspect, the polypeptide is a Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia spededonium, Thielavia setosa, Thielaviasubthermophila, Thielavia terrestris, Thielavia terricola, Thielaviathermophila, Thielavia variospora, or Thielavia wareingii polypeptidehaving endoglucanase activity.

In a more preferred aspect, the polypeptide is a Thielavia terrestrispolypeptide having endoglucanase activity, and most preferably aThielavia terrestris NRRL 8126 polypeptide having endoglucanaseactivity, e.g., the polypeptide of SEQ ID NO: 2 or the maturepolypeptide thereof.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising or consisting of a nucleotide sequence which encode apolypeptide of the present invention having endoglucanase activity.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pTter7F whichis contained in E. coli NRRL B-30837. In another preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region of SEQ ID NO: 1. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 52 to 1008 ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding region containedin plasmid pTter7F which is contained in E. coli NRRL B-30837. Thepresent invention also encompasses nucleotide sequences which encode apolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 2 or the mature polypeptide thereof, which differ from SEQ ID NO:1 or the mature polypeptide coding sequence thereof by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 1 which encode fragments of SEQ ID NO: 2 thathave endoglucanase activity.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 1, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 2. In a preferred aspect, the maturepolypeptide is amino acids 18 to 336 of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Thielavia, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides comprisingnucleotide sequences which have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 60%, preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 52 to 1008 of SEQ ID NO: 1.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forendoglucanase activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-enzyme interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labeling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) a complementary strand of (i) or (ii); or allelic variantsand subsequences thereof (Sambrook et al., 1989, supra), as definedherein. In a preferred aspect, the mature polypeptide coding sequence isnucleotides 52 to 1008 of SEQ ID NO: 1.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) a complementary strand of (i) or (ii); and (b) isolating thehybridizing polynucleotide, which encodes a polypeptide havingendoglucanase activity. In a preferred aspect, the mature polypeptidecoding sequence is nucleotides 52 to 1008 of SEQ ID NO: 1.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice, i.e.,secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

In a preferred aspect, the signal peptide is amino acids 1 to 17 of SEQID NO: 2. In another preferred aspect, the signal peptide coding regionis nucleotides 1 to 51 of SEQ ID NO: 1 which encode amino acids 1 to 17of SEQ ID NO: 2.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMAL and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Thielavia terricola,Thielavia thermophila, Thielavia variospora, Thielavia wareingii,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating acell, which in its wild-type form is capable of producing thepolypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. In a preferred aspect,the cell is of the genus Thielavia. In a more preferred aspect, the cellis Thielavia terrestris. In a most preferred aspect, the cell isThielavia terrestris NRRL 8126.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 1, wherein the mutant nucleotide sequence encodes a polypeptidewhich consists of the mature polypeptide of SEQ ID NO: 2, and (b)recovering the polypeptide. In a preferred aspect, the maturepolypeptide is amino acids 18 to 336 of SEQ ID NO: 2.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having endoglucanase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome or chloroplastgenome and propagating the resulting modified plant or plant cell into atransgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having endoglucanase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Endoglucanase Activity

The present invention also relates to methods for producing a mutant ofa parent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or morenucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleotidesequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence which is then transformed into theparent cell to produce a defective gene. By homologous recombination,the defective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous polypeptides. Therefore, the present invention furtherrelates to methods for producing a homologous or heterologouspolypeptide comprising: (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of endoglucanase activityby fermentation of a cell which produces both a polypeptide of thepresent invention as well as the protein product of interest by addingan effective amount of an agent capable of inhibiting endoglucanaseactivity to the fermentation broth before, during, or after thefermentation has been completed, recovering the product of interest fromthe fermentation broth, and optionally subjecting the recovered productto further purification.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of endoglucanase activityby cultivating the cell under conditions permitting the expression ofthe product, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the endoglucanase activitysubstantially, and recovering the product from the culture broth.Alternatively, the combined pH and temperature treatment may beperformed on an enzyme preparation recovered from the culture broth. Thecombined pH and temperature treatment may optionally be used incombination with a treatment with an endoglucanase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the endoglucanase activity. Complete removal of endoglucanaseactivity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-3 or 10-11 and a temperature in the range of atleast 75-85° C. for a sufficient period of time to attain the desiredeffect, where typically, 1 to 3 hours is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyendoglucanase-free product is of particular interest in the productionof eukaryotic polypeptides, in particular fungal proteins such asenzymes. The enzyme may be selected from, e.g., an amylolytic enzyme,lipolytic enzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase,or plant cell-wall degrading enzyme. Examples of such enzymes include anaminopeptidase, amylase, amyloglucosidase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Theendoglucanase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from endoglucanase activity which is produced by amethod of the present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theendoglucanase activity of the composition has been increased, e.g., withan enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger,or Aspergillus oryzae; Fusarium, preferably Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusariumtoruloseum, Fusarium trichothecioides, or Fusarium venenatum; Humicola,preferably Humicola insolens or Humicola lanuginosa; or Trichoderma,preferably Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having endoglucanase activity, or compositions thereof.

Degradation of Biomass to Monosaccharides, Disaccharides, andPolysaccharides

The polypeptides having endoglucanase activity, and host cells of thepresent invention, may be used in the production of monosaccharides,disaccharides, and polysaccharides as chemical or fermentationfeedstocks from biomass for the production of ethanol, plastics, orother products or intermediates. The polypeptides having endoglucanaseactivity may be in the form of a crude fermentation broth with orwithout the cells removed or in the form of a semi-purified or purifiedenzyme preparation. Alternatively, a host cell of the present inventionmay be used as a source of the polypeptide having endoglucanase activityin a fermentation process with the biomass.

Biomass can include, but is not limited to, wood resources, municipalsolid waste, wastepaper, and crop residues (see, for example, Wiselogelet al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork).

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened throughpolymeric lignin covalently cross-linked to hemicellulose. Cellulose isa homopolymer of anhydrocellobiose and thus a linearbeta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which helps stabilize the cell wall matrix.

Three major classes of glycohydrolases are used to breakdown cellulosicbiomass:

-   -   (1) The “endo-1,4-beta-glucanases” or        1,4-beta-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act        randomly on soluble and insoluble 1,4-beta-glucan substrates.    -   (2) The “exo-1,4-beta-D-glucanases” including both the        1,4-beta-D-glucan glucohydrolases (EC 3.2.1.74), which liberate        D-glucose from 1,4-beta-D-glucans and hydrolyze D-cellobiose        slowly, and cellobiohydrolases (1,4-beta-D-glucan        cellobiohydrolases, EC 3.2.1.91), which liberate D-cellobiose        from 1,4-beta-glucans.    -   (3) The “beta-D-glucosidases” or beta-D-glucoside        glucohydrolases (EC 3.2.1.21), which act to release D-glucose        units from cellobiose and soluble cellodextrins, as well as an        array of glycosides.

These three classes of enzymes work together synergistically resultingin efficient decrystallization and hydrolysis of native cellulose frombiomass to yield reducing sugars.

The polypeptides having endoglucanase activity of the present inventionmay be used in conjunction with the above-noted enzymes to furtherdegrade the cellulose component of the biomass substrate, (see, forexample, Brigham et al., 1995, in Handbook on Bioethanol (Charles E.Wyman, editor), pp. 119-141, Taylor & Francis, Washington D.C.; Lee,1997, Journal of Biotechnology 56: 1-24).

Ethanol can be produced by enzymatic degradation of biomass andconversion of the released saccharides to ethanol. This kind of ethanolis often referred to as bioethanol or biofuel. It can be used as a fueladditive or extender in blends of from less than 1% and up to 100% (afuel substitute).

Detergent Compositions

The polypeptides having endoglucanase activity of the present inventionmay be added to and thus become a component of a detergent composition.

The detergent composition of the present invention may be, for example,formulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or formulated asa detergent composition for use in general household hard surfacecleaning operations, or formulated for hand or machine dishwashingoperations.

In a specific aspect, the present invention provides a detergentadditive comprising the polypeptides having endoglucanase activity ofthe present invention. The detergent additive as well as the detergentcomposition may comprise one or more other enzymes such as a protease,lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase,mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase,and/or peroxidase.

In general the properties of the enzymatic components should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzymatic components should be present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metalloprotease, preferably an alkaline microbial proteaseor a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™, Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OXP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta,1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipases include Lipolase™, Lipex™, andLipolase Ultra™ (Novozymes A/S).

Amylases: Suitable amylases (α and/or β) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g., a special strain of Bacillus licheniformis, described inmore detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, or Trichodermae.g., the fungal cellulases produced from Humicola insolens,Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat.No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,776,757, and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include Celluclast®, Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The enzymatic component(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the present invention, i.e., a separate additive or a combinedadditive, can be formulated, for example, as a granulate, liquid,slurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the present invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers,and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system that may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzymatic component(s) of the detergent composition of the presentinvention may be stabilized using conventional stabilizing agents, e.g.,a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol,lactic acid, boric acid, or a boric acid derivative, e.g., an aromaticborate ester, or a phenyl boronic acid derivative such as 4-formylphenylboronic acid, and the composition may be formulated as described in, forexample, WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions any enzymatic component, in particular thepolypeptides having endoglucanase activity of the present invention, maybe added in an amount corresponding to 0.01-100 mg of enzyme protein perliter of wash liquor, preferably 0.05-5 mg of enzyme protein per literof wash liquor, in particular 0.1-1 mg of enzyme protein per liter ofwash liquor.

The polypeptides having endoglucanase activity of the present inventionmay additionally be incorporated in the detergent formulations disclosedin WO 97/07202 which is hereby incorporated as reference.

Signal Peptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to a nucleotide sequenceencoding a signal peptide comprising or consisting of amino acids 1 to17 of SEQ ID NO: 2, which allows secretion of the protein into a culturemedium, wherein the gene is foreign to the nucleotide sequence.

In a preferred aspect, the nucleotide sequence comprises nucleotides 1to 51 of SEQ ID NO: 1. In another preferred aspect, the nucleotidesequence consists of nucleotides 1 to 51 of SEQ ID NO: 1.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising: (a) cultivating such a recombinant host cell underconditions suitable for production of the protein; and (b) recoveringthe protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

SDS-PAGE gels, loading buffer, and running buffer were obtained fromInvitrogen/Novex (Carlsbad, Calif.). Sequencing grade modified trypsinwas from Princeton Separations (Aldelphia, N.J.). BioSafe Commassie BlueG250 protein stains were obtained from BioRad Laboratories (Hercules,Calif.).

Strains

Aspergillus oryzae Jal250 strain (WO 99/61651) was used for expressionof a Thielavia terrestris polypeptide having endoglucanase activity.Thielavia terrestris NRRL strain 8126 was used as the source of a genefor a Family 7F polypeptide having endoglucanase activity.

Media

PDA plates were composed per liter of 39 grams of potato dextrose agar.

NNCYP medium was composed per liter of 5.0 g of NH₄NO₃, 0.5 g ofMgSO₄.7H₂O, 0.3 g of CaCl₂, 2.5 g of citric acid, 1.0 g of BactoPeptone, 5.0 g of yeast extract, 1 ml of COVE trace metals, andsufficient K₂HPO₄ to achieve a final pH of approximately 5.4.

NNCYPmod medium was composed per liter of 1.0 g of NaCl, 5.0 g ofNH₄NO₃, 0.2 g of MgSO₄.7H₂O, 0.2 g of CaCl₂, 2.0 g of citric acid, 1.0 gof Bacto Peptone, 5.0 g of yeast extract, 1 ml of COVE trace metalssolution, and sufficient K₂HPO₄ to achieve the final pH of approximately5.4.

Cove trace metals solution was composed per liter of 0.04 g ofNa₂B₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

LB plates were composed per liter of 10 g of tryptone, 5 g of yeastextract, 5 g of sodium chloride, and 15 g of Bacto Agar.

MDU2BP medium was composed per liter of 45 g of maltose, 1 g ofMgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂HSO₄, 12 g of KH₂PO₄, 2 g of urea, and500 μl of AMG trace metals, the pH was adjusted to 5.0 and then filtersterilized with a 0.22 μm filtering unit.

AMG trace metals was composed per liter of 14.3 g of ZnSO₄.7H₂O, 2.5 gof CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.7H₂O, and 3 g of citric acid.

SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl,2.5 mM KCl, 10 mM MgCl₂, and 10 mM MgSO₄, sterilized by autoclaving andthen added filter-sterilized glucose to 20 mM.

Freezing medium was composed of 60% SOC and 40% glycerol.

2×YT medium was composed per liter of 16 g of tryptone, 10 g of yeastextract, 5 g of NaCl, and 15 g of Bacto agar, sterilize by autoclaving.

Example 1 Expressed Sequence Tags (EST) cDNA Library Construction

Thielavia terrestris NRRL 8126 was cultivated in 50 ml of NNCYPmodmedium supplemented with 1% glucose in a 250 ml flask at 45° C., 200 rpmfor 24 hours. A two ml aliquot from the 24 hour liquid culture was usedto seed a 500 ml flask containing 100 ml of NNCYPmod medium supplementedwith 2% Sigmacell-20 (Sigma Chemical Co., St. Louis, Mo.). The culturewas incubated at 45° C., 200 rpm for 3 days. The mycelia were harvestedby filtration through a Buchner funnel with a glass fiber prefilter(Nalgene, Rochester N.Y.), washed twice with 10 mM Tris-HCl-1 mM EDTA pH8 (TE), and quick frozen in liquid nitrogen.

Total RNA was isolated using the following method. Frozen mycelia ofThielavia terrestris NRRL 8126 were ground in an electric coffeegrinder. The ground material was mixed 1:1 v/v with 20 ml of Fenazol(Ambion, Inc., Austin, Tex.) in a 50 ml Falcon tube. Once the myceliawere suspended, they were extracted with chloroform and three times witha mixture of phenol-chloroform-isoamyl alcohol 25:24:1 v/v/v. From theresulting aqueous phase, the RNA was precipitated by adding 1/10 volumeof 3 M sodium acetate pH 5.2 and 1.25 volumes of isopropanol. Theprecipitated RNA was recovered by centrifugation at 12,000×g for 30minutes at 4° C. The final pellet was washed with cold 70% ethanol, airdried, and resuspended in 500 ml of diethylpyrocarbonate-treated water(DEPC-water).

The quality and quantity of the purified RNA was assessed with anAgilent Bioanalyzer 2100 (Agilent Technologies, Inc., Palo Alto,Calif.). Polyadenylated mRNA was isolated from 360 μg of total RNA withthe aid of a Poly (A) Purist Magnetic Kit (Ambion, Inc., Austin, Tex.)according to the manufacturer's instructions.

To create the cDNA library, a CloneMiner™ Kit (Invitrogen, Carlsbad,Calif.) was employed to construct a directional library that does notrequire the use of restriction enzyme cloning, thereby reducing thenumber of chimeric clones and size bias.

To insure successful first strand synthesis of the cDNA, two reactionswere performed in parallel with two different concentrations of mRNA(2.2 and 4.4 μg of poly(A)+ mRNA). The mRNA samples were mixed with aBiotin-attB2-Oligo(dt) primer (CloneMiner™ Kit, Invitrogen, Carlsbad,Calif.), 1× first strand buffer (Invitrogen, Carlsbad, Calif.), 2 μl of0.1 M dithiothreitol (DTT), 10 mM of each dNTP, and water to a finalvolume of 18 and 16 μl, respectively. The reaction mixtures were mixedcarefully and then 2 and 4 μl of SuperScript™ reverse transcriptase(Invitrogen, Carlsbad, Calif.) were added and incubated at 45° C. for 60minutes to synthesize the first complementary strand.

For second strand synthesis, to each first strand reaction was added 30μl of 5× second strand buffer (Invitrogen, Carlsbad, Calif.), 3 μl of 10mM of each dNTP, 10 units of E. coli DNA ligase (Invitrogen, Carlsbad,Calif.), 40 units of E. coli DNA polymerase I (Invitrogen, Carlsbad,Calif.), and 2 units of E. coli RNase H (Invitrogen, Carlsbad, Calif.)in a total volume of 150 μl. The mixtures were then incubated at 16° C.for two hours. After the two-hour incubation, 2 μl of T4 DNA polymerase(Invitrogen, Carlsbad, Calif.) were added to each reaction and incubatedat 16° C. for 5 minutes to create a bunt-ended cDNA. The cDNA reactionswere extracted with a mixture of phenol-chloroform-isoamyl alcohol25:24:1 v/v/v and precipitated in the presence of 20 μg of glycogen, 120μl of 5 M ammonium acetate, and 660 μl of ethanol. After centrifugationat 12,000×g for 30 minutes at 4° C., the cDNA pellets were washed withcold 70% ethanol, dried under vacuum for 2-3 minutes, and resuspended in18 μl of DEPC-water. To each resuspended cDNA sample was added 10 μl of5× adapted buffer (Invitrogen, Carlsbad, Calif.), 10 μg of attB1 adapter(Invitrogen, Carlsbad, Calif.) shown below, 7 μl of 0.1 M DTT, and 5units of T4 DNA ligase (Invitrogen, Carlsbad, Calif.).

attB1 adapter top strand: (SEQ ID NO: 3)5′-TCGTCGGGGACAACTTTGTACAAAAAAGTTGG-3′ attB1 adapter bottom strand: (SEQID NO: 4) 3′-CCCCTGTTGAAACATGTTTTTTCAACCp-5′

Ligation reactions were incubated overnight at 16° C. Excess adapterswere removed by size-exclusion chromatography in 1 ml of Sephacryl™S-500 HR resin (Amersham Biosciences, Piscataway, N.J.). Columnfractions were collected according to the CloneMiner™ Kit's instructionsand fractions 3 to 14 were analyzed with an Agilent Bioanalyzer todetermine the fraction at which the attB1 adapters started to elute.This analysis showed that the adapters started eluting around fraction10 or 11. For the first library fractions 6 to 11 were pooled and forthe second library fractions 4-11 were pooled.

Cloning of the cDNA was performed by homologous DNA recombinationaccording to the Gateway System protocol (Invitrogen, Carlsbad, Calif.)using BP Clonase™ (Invitrogen, Carlsbad, Calif.) as the recombinase.Each BP Clonase™ recombination reaction contained approximately 70 ng ofattB-flanked-cDNA, 250 ng of pDONR™222, 2 μl of 5×BP Clonase™ buffer, 2μl of TE buffer, and 3 μl of BP Clonase™. All reagents were obtainedfrom Invitrogen, Carlsbad, Calif. Recombination reactions were incubatedat 25° C. overnight.

Heat-inactivated BP recombination reactions were then divided in 6aliquots and electroporated into ElectroMax™ DH10B electrocompetentcells (Invitrogen, Carlsbad, Calif.) using a BioRad Gene Pulser II(BioRad, Hercules, Calif.) with the following parameters: Voltage: 2.0kV; Resistance: 200Ω; and Capacity: 25 pF. Electroporated cells wereresuspended in 1 ml of SOC medium and incubated at 37° C. for 60 minuteswith constant shaking at 200 rpm. After the incubation period, thetransformed cells were pooled and mixed 1:1 with freezing medium. A 200μl aliquot was removed for library titration and then the rest of eachlibrary was aliquoted into 1.8 ml cryovials (Wheaton Science Products,Millville, N.J.) and stored frozen at −80° C.

Four serial dilutions of each library were prepared: 1/100, 1/1000,1/10⁴, 1/10⁵. From each dilution 100 μl were plated onto 150 mm LBplates supplemented with 50 μg of kanamycin per ml and incubated at 37°C. overnight. The number of colonies on each dilution plate were countedand used to calculate the total number of transformants in each library.

The first library was shown to have 5.4 million independent clones andthe second library was show to have 9 million independent clones.

Example 2 Template Preparation and Nucleotide Sequencing of cDNA Clones

Aliquots from both libraries were mixed and plated onto 25×25 cm LBplates supplemented with 50 μg of kanamycin per ml. Individual colonieswere arrayed onto 96-well plates containing 100 μl of LB supplementedwith 50 μg of kanamycin per ml with the aid of a Genetix QPix Robot(Genetix Inc., Boston, Mass.). Forty five 96-well plates were obtainedfor a total of 4320 individual clones. The plates were incubatedovernight at 37° C. with shaking at 200 rpm. After incubation, 100 μl ofsterile 50% glycerol was added to each well. The transformants werereplicated with the aid of a 96-pin tool (Boekel, Feasterville, Pa.)into secondary, deep-dish 96-well microculture plates (Advanced GeneticTechnologies Corporation, Gaithersburg, Md.) containing 1 ml ofMagnificent Broth™ (MacConnell Research, San Diego, Calif.) supplementedwith 50 μg of kanamycin per ml in each well. The primary microtiterplates were stored frozen at −80° C. The secondary deep-dish plates wereincubated at 37° C. overnight with vigorous agitation at 300 rpm on arotary shaker. To prevent spilling and cross-contamination, and to allowsufficient aeration, each secondary culture plate was covered with apolypropylene pad (Advanced Genetic Technologies Corporation,Gaithersburg, Md.) and a plastic microtiter dish cover. Plasmid DNA wasprepared with a MWG Robot-Smart 384 (MWG Biotech Inc., High Point, N.C.)and Montage Plasmid Miniprep Kits (Millipore, Billerica, Mass.).

Sequencing reactions were performed using Big-Dye™ (Applied Biosystems,Inc., Foster City, Calif.) terminator chemistry (Giesecke et al., 1992,Journal of Virology Methods 38: 47-60) and a M13 Forward (−20)sequencing primer shown below.

5′-GTAAAACGACGGCCAG-3′ (SEQ ID NO: 5)

The sequencing reactions were performed in a 384-well format with aRobot-Smart 384 (MWG Biotech Inc., High Point, N.C.) and terminatorremoval with Millipore MultiScreen Seq384 Sequencing Clean-up Kits(Millipore, Billerica, Mass.). Reactions contained 6 μl of plasmid DNAand 4 μl of sequencing master-mix containing 2 μl of 5× sequencingbuffer (Millipore, Billerica, Mass.), 1 μl of Big-Dye™ terminator(Applied Biosystems, Inc., Foster City, Calif.), 1.6 pmoles of M13Forward primer, and 1 μl of water. Single-pass DNA sequencing wasperformed with an ABI PRISM Automated DNA Sequencer Model 3700 (AppliedBiosystems, Foster City, Calif.).

Example 3 Analysis of DNA Sequence Data of cDNA Clones

Base calling, quality value assignment, and vector trimming wereperformed with the assistance of PHRED/PHRAP software (University ofWashington, Seattle, Wash.). Clustering analysis of the ESTs wasperformed with a Parcel Transcript Assembler v. 2.6.2. (Paracel, Inc.,Pasadena, Calif.). Analysis of the EST clustering indicated 395independent clusters.

Sequence homology analysis of the assembled EST sequences against thePIR database was performed with the Blastx program (Altschul et. al.,1990, J. Mol. Biol. 215: 403-410) on a 32-node Linux cluster (Paracel,Inc., Pasadena, Calif.) using the BLOSUM 62 matrix (Henikoff, 1992,Proc. Natl. Acad. Sci. USA 89: 10915-10919) From the 395 clusters, 246had blast hits to known genes in public protein databases and 149 had nosignificant hits against these databases. Among these 246 genes, 13 hadhits against well characterized homologues of glycosyl hydrolase genes.

Example 4 Identification of cDNA Clones Encoding a Family 7Endoglucanase (CEL7F)

A cDNA clone encoding a Family 7 endoglucanase (CEL7F) was initiallyidentified by its identity to the Family 7 endoglucanase EG-1 proteinfrom Trichoderma longibrachiatum (NREF NF00756647). This analysisindicated that the two proteins were 44% identical at the protein levelover a 113 amino acid (339 basepairs) stretch. After this initialidentification clone Tter08C4 was retrieved from the original frozenstock plate and streaked onto a LB plate supplemented with 50 μg ofkanamycin per ml. The plate was incubated overnight at 37° C. and thenext day a single colony from the plate was used to inoculate 3 ml of LBsupplemented with 50 μg of kanamycin per ml. The liquid culture wasincubated overnight at 37° C. and plasmid DNA was prepared with aBioRobot 9600 (QIAGEN, Inc., Valencia, Calif.). Clone Tter08C4 plasmidDNA was sequenced again with Big-Dye™ terminator chemistry as describedabove, using the M13 forward and a Poly-T primer shown below to sequencethe 3′ end of the clone.

5′-TTTTTTTTTTTTTTTTTTTTTTTVN-3′ (SEQ ID NO: 6)

Where V=G, A, C and N=G, A, C, T

Blastx homology analysis of the sequence information indicated that theprotein encoded by clone Tter08C4 was similar to the Trichoderma reeseiEG1 protein (NREF NF00494331). These proteins were 46% identical over a365 amino acid stretch.

Analysis of the deduced protein sequence of clone Tter08C4 with theInterproscan program (Zdobnov and Apweiler, 2001, Bioinformatics 17:847-8) showed that the gene contained the sequence signature of theFamily 7 proteins. This sequence signature known as the Pfam patternPF00840 (Bateman et al., 2002, Nucleic Acids Research 30: 276-280) wasfound 18 amino acids from the starting amino acid methionine confirmingthat clone Tter08C4 encodes a Family 7 endoglucanase.

The cDNA sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ IDNO: 2) of the Thielavia terrestris endoglucanase are shown in FIGS. 1Aand 1B. The cDNA clone encodes a polypeptide of 336 amino acids. The %G+C content of the cDNA clone of the gene is 67.5% and of the matureprotein coding region (nucleotides 55 to 1011 of SEQ ID NO: 1) is 67.5%.Using the SignalP software program (Nielsen et al., 1997, ProteinEngineering 10:1-6), a signal peptide of 17 residues was predicted. Thepredicted mature protein contains 319 amino acids with a molecular massof 33.3 kDa.

A comparative alignment of endoglucanase Family 7 sequences wasdetermined by the Clustal W method (Higgins, 1989, supra) using theLASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with anidentity table and the following multiple alignment parameters: Gappenalty of 10 and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, windows=5, and diagonals=5. Thealignment showed that the deduced amino acid sequence of the matureThielavia terrestris CEL7F gene shares 46.7% identity to the deducedamino acid sequence of the catalytic region of the Trichoderma reeseiendoglucanase I gene (NREF NF00494331, Uniprot Q5BMS5) and 44.9%identity to the deduced amino acid sequence of the full-lengthTrichoderma reesei endoglucanase I gene (NREF NF00494331, UniprotQ5BMS5). Analysis of the alignment of the catalytic regions of theseproteins showed that the Thielavia terrestris CEL7F endoglucanase ismissing at least three discrete sequence motifs that are conserved inall other known members of glycosyl hydrolase Family 7 from fungi. Twoof these consist of more than ten amino acid residues and contain highlyconserved residues that are not present in the Thielavia terrestrisCEL7F endoglucanase.

Once the identity of clone Tter08C4 was confirmed, a 0.5 μl aliquot ofplasmid DNA from this clone, designated pTter7F (FIG. 2), wastransferred into a vial of E. coli TOP10 cells (Invitrogen, Carlsbad,Calif.), gently mixed, and incubated on ice for 10 minutes. The cellswere then heat-shocked at 42° C. for 30 seconds and incubated again onice for 2 minutes. The cells were resuspended in 250 μl of SOC mediumand incubated at 37° C. for 60 minutes with constant shaking (200 rpm).After the incubation period, two 30 μl aliquots were plated onto LBplates supplemented with 50 μg of kanamycin per ml and incubatedovernight at 37° C. The next day a single colony was picked and streakedonto three 1.8 ml cryovials containing about 1.5 ml of LB agarosesupplemented with 50 μg of kanamycin per ml. The vials were sealed withPetriSeal™ (Diversified Biotech, Boston Mass.) and deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30802, with a deposit date of Apr. 11, 2005.

Example 5 Construction of pAILo2 Expression Vector

Expression vector pAILo1 was constructed by modifying pBANe6 (U.S. Pat.No. 6,461,837), which comprises a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase (NA2-tpi promoter), Aspergillus nigeramyloglucosidase terminator sequence (AMG terminator), and Aspergillusnidulans acetamidase gene (amdS). All mutagenesis steps were verified bysequencing using Big-Dye™ terminator chemistry as described.Modification of pBANe6 was performed by first eliminating three Nco Irestriction sites at positions 2051, 2722, and 3397 bp from the amdSselection marker by site-directed mutagenesis. All changes were designedto be “silent” leaving the actual protein sequence of the amdS geneproduct unchanged. Removal of these three sites was performedsimultaneously with a GeneEditor™ in vitro Site-Directed Mutagenesis Kit(Promega, Madison, Wis.) according to the manufacturer's instructionsusing the following primers (underlined nucleotide represents thechanged base):

AMDS3NcoMut (2050): 5′-GTGCCCCATGATACGCCTCCGG-3′ (SEQ ID NO: 7)AMDS2NcoMut (2721): 5′-GAGTCGTATTTCCAAGGCTCCTGACC-3′ (SEQ ID NO: 8)AMDS1NcoMut (3396): 5′-GGAGGCCATGAAGTGGACCAACGG-3′ (SEQ ID NO: 9)

A plasmid comprising all three expected sequence changes was thensubmitted to site-directed mutagenesis, using a QuickChange™Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.), toeliminate the Nco I restriction site at the end of the AMG terminator atposition 1643. The following primers (underlined nucleotide representsthe changed base) were used for mutagenesis:

Upper Primer to mutagenize the AMG terminator sequence: (SEQ ID NO: 10)5′-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG- 3′ Lower Primer tomutagenize the AMG terminator sequence: (SEQ ID NO: 11)5′-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG- 3′

The last step in the modification of pBANe6 was the addition of a newNco I restriction site at the beginning of the polylinker using aQuickChange™ Site-Directed Mutagenesis Kit and the following primers(underlined nucleotides represent the changed bases) to yield pAILo1(FIG. 3).

Upper Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 12)5′-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3′ Lower Primer tomutagenize the NA2-tpi promoter: (SEQ ID NO: 13)5′-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3′

The amdS gene of pAILo1 was swapped with the Aspergillus nidulans pyrGgene. Plasmid pBANe10 (FIG. 4) was used as a source for the pyrG gene asa selection marker. Analysis of the sequence of pBANe10 showed that thepyrG marker was contained within an Nsi I restriction fragment and doesnot contain either Nco I or Pac I restriction sites. Since the amdS isalso flanked by Nsi I restriction sites the strategy to switch theselection marker was a simple swap of Nsi I restriction fragments.Plasmid DNA from pAILo1 and pBANe10 were digested with the restrictionenzyme Nsi I and the products purified by agarose gel electrophoresis.The Nsi I fragment from pBANe10 containing the pyrG gene was ligated tothe backbone of pAILo1 to replace the original Nsi I DNA fragmentcontaining the amdS gene. Recombinant clones were analyzed byrestriction digest to determine that they had the correct insert andalso its orientation. A clone with the pyrG gene transcribed in thecounterclockwise direction was selected. The new plasmid was designatedpAILo2 (FIG. 5).

Example 6 Cloning of the Family CEL7F Endoglucanase Gene into anAspergillus oryzae Expression Vector

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the full-length open reading frame from Thielavia terrestris ESTTter08C4 encoding a Family CEL7F endoglucanase. An In-Fusion Cloning Kit(BD Biosciences, Palo Alto, Calif.) was used to clone the fragmentdirectly into pAILo2.

In-Fusion Forward primer: (SEQ ID NO: 14)5′-ACTGGATTACCATGACCCTACGGCTCCCTGTCATCA-3′ In-Fusion Reverse primer:(SEQ ID NO: 15) 5′-TCACCTCTAGTTAATTAACTAGTTCTTCGTGGTAGACC-3′Bold letters represent coding sequence. The remaining sequence containssequence identity compared with the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 50 ng of pTter11C9 DNA, 1× Pfx Amplification Buffer(Invitrogen, Carlsbad, Calif.), 6 μl of 10 mM blend of dATP, dTTP, dGTP,and dCTP, 2.5 units of Platinum Pfx DNA Polymerase (Invitrogen,Carlsbad, Calif.), 1 μl of 50 mM MgSO₄, and 5 μl of 10× pCRx Enhancersolution (Invitrogen, Carlsbad, Calif.) in a final volume of 50 μl. AnEppendorf Mastercycler 5333 (Eppendorf Scientific, Inc., Westbury, N.Y.)was used to amplify the fragment programmed for one cycle at 98° C. for2 minutes; and 35 cycles each at 94° C. for 30 seconds, 65° C. for 30seconds, and 68° C. for 1.5 minutes. After the 35 cycles, the reactionwas incubated at 68° C. for 10 minutes and then cooled at 10° C. untilfurther processed. A 1.4 kb PCR reaction product was isolated on a 0.8%GTG-agarose gel (Cambrex Bioproducts One Meadowlands Plaza EastRutherford, N.J. 07073) using 40 mM Tris base-20 mM sodium acetate-1 mMdisodium EDTA (TAE) buffer and 0.1 μg of ethidium bromide per ml. TheDNA band was visualized with the aid of a Dark Reader™ (Clare ChemicalResearch, Dolores, Colo.) to avoid UV-induced mutations. The 1.4 kb DNAband was excised with a disposable razor blade and purified with anUltrafree-DA spin cup (Millipore, Billerica, Mass.) according to themanufacturer's instructions.

The vector pAILo2 was linearized by digestion with Nco I and Pac 1. Thefragment was purified by gel electrophoresis and ultrafiltration asdescribed above. Cloning of the purified PCR fragment into thelinearized and purified pAILo2 vector was performed with an In-FusionCloning Kit (BD Biosciences, Palo Alto, Calif.). The reaction (20 μl)contained 1× In-Fusion Buffer (BD Biosciences, Palo Alto, Calif.), 1×BSA(BD Biosciences, Palo Alto, Calif.), 1 μl of In-Fusion enzyme (diluted1:10) (BD Biosciences, Palo Alto, Calif.), 100 ng of pAILo2 digestedwith Nco I and Pac I, and 50 ng of the Thielavia terrestris CEL7Fpurified PCR product. The reaction was incubated at room temperature for30 minutes. A 2 μl sample of the reaction was used to transform E. coliXL10 SoloPac® Gold cells (Stratagene, La Jolla, Calif.) according to themanufacturer's instructions. After the recovery period, two 100 μlaliquots from the transformation reaction were plated onto 150 mm 2×YTplates supplemented with 100 μg of ampicillin per ml. The plates wereincubated overnight at 37° C. A set of eight putative recombinant cloneswas selected at random from the selection plates and plasmid DNA wasprepared from each one using a BioRobot 9600. Clones were analyzed byXho I restriction digest. Two clones that had the expected restrictiondigest pattern were then sequenced to confirm that there were nomutations in the cloned insert. Clone #1 was selected and designatedpAILo22 (FIG. 6).

Example 7 Expression of the Thielavia terrestris Family CEL7FEndoglucanase Gene in Aspergillus oryzae JAL250

Aspergillus oryzae Jal250 (WO 99/61651) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422. Five micrograms of pAILo22 (as well as pAILo2 as a vectorcontrol) were used to transform Aspergillus oryzae JAL250 protoplasts.

The transformation of Aspergillus oryzae Jal250 with pAILo2 yieldedabout 50 transformants. Eight transformants were isolated to individualPDA plates and incubated for five days at 34° C.

Confluent spore plates were washed with 5 ml of 0.01% Tween 80 and thespore suspension was used to inoculate 25 ml of MDU2BP medium in 125 mlglass shake flasks. Transformant cultures were incubated at 34° C. withconstant shaking at 200 rpm. At day five post-inoculation, cultures werecentrifuged at 6000×g and their supernatants collected. Five μl of eachsupernatant were mixed with an equal volume of 2× loading buffer (10%1-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-Glycine SDS-PAGEgel and stained with Simply Blue SafeStain (Invitrogen, Carlsbad,Calif.). SDS-PAGE profiles of the culture broths showed that six out ofeight transformants had a new protein band of approximately 40 kDa.Transformant number 7 was selected for further studies and designatedAspergillus oryzae Jal250AILo22.

Example 8 Large Shake Flask Cultures of Aspergillus oryzae Jal250AILo22

Aspergillus oryzae Jal250AILo22 spores were spread onto a PDA plate andincubated for five days at 34° C. The confluent spore plate was washedtwice with 5 ml of 0.01% Tween 80 to maximize the number of sporescollected. The spore suspension was then used to inoculate 500 ml ofMDU2BP medium in a two-liter Fernbach flask. The transformant culturewas incubated at 34° C. with constant shaking (200 rpm). At day fivepost-inoculation, the culture broth was collected by filtration on a 500ml, 75 mm Nylon filter unit with a pore size of 0.45 μm with aglass-fiber pre-filter. A 5 μl sample of the broth was analyzed bySDS-PAGE as described above to confirm that the protein pattern was thesame as the one obtained before. Once the broth was shown to contain the40 kDa protein band, the broth was submitted to enzymaticcharacterization.

Example 9 Characterization of the Thielavia terrestris CEL7FEndoglucanase

The Aspergillus oryzae Jal250AILo22 broth described in Example 8 wasfiltered through a 0.22 μm pore-size filter (Millipore, Billerica,Mass.), concentrated using an Amicon stirred cell equipped with a PM10membrane (Millipore, Billerica, Mass.), and desalted using an Econo-Pac10DG column (BioRad Laboratories, Hercules, Calif.).

Broth from Aspergillus oryzae Jal250 (vector alone), as a negativecontrol, was treated the same as above.

Dyed substrates used to evalulate the substrate specificity of theThielavia terrestris CEL7F endoglucanase included: AZCL-arabinoxylan(wheat), AZCL-β-glucan, AZCL-dextran, AZCL-HE-cellulose, AZCL-potatogalactan, AZCL-galactomannan (Carob), AZCL-xylan (Birchwood),AZCL-xyloglucan (Megazyme, Bray, Ireland), and Chitin Azure (Sigma, StLouis, Mo.).

The activity assays were performed in 96-deep-well plates (AxygenScientific, Union City, Calif.) sealed by a plate sealer (ALPS-300,Abgene, Epsom, UK). Eight hundred μl of the above substrates (6.25 g perliter of 50 mM sodium acetate pH 5.0) were transferred into each well ofthe 96-deep-well-plate, followed by 180 μl of 50 mM sodium acetate pH5.0 and 20 μl of Thielavia terrestris CEL7F endoglucanase solution (0.25g/L) to start the reactions. The substrate concentration and enzymeloading in the final reaction mixture were 5 g per liter and 1 mg ofenzyme per g of substrate, respectively. Aspergillus oryzae Jal250 brothwas tested along with Thielavia terrestris CEL7F endoglucanase brothunder the same condition, serving as a negative control. The reactionswere incubated at 50° C. without mixing. Before sampling, the deep wellplates were centrifuged in a plate centrifuge (Sorvall RT7, GlobalMedical Instrumentation, Ramsey, Minn.) at 3000 rpm for 5 minutes. A 150μl sample of the supernatant was transferred into a 96-well filtrationplate (0.45 μm pore size, Millipore, Billerica, Mass.), vacuumed andfiltrate was collected. A 100 μl sample of the filtrate was transferredinto another 96-well plate and the absorbance at 590 nm was measuredusing a Spectra MAX340 (Molecular Devices, Sunnyvale, Calif.).

After 1 hour and 92 hour incubations, the dye released from thedifferent dyed substrates by Thielavia terrestris CEL7F endoglucanase(after subtracting the dye released by Aspergillus oryzae Jal250) areshown in Table 1 as relative 590 nm values.

TABLE 1 Relative A_(590 nm) after incubating Thielavia terrestrisendoglucanase with different dyed substrates for 1 hour and 92 hours at50° C., pH 5.0. Time Chitin hr AX βG Dex HEC Gal GM Xly XG Azure  1 0.000.22 0.00 0.16 0.00 0.00 0.01 0.01 0.00 92 0.58 0.78 0.00 1.00 0.00 0.080.32 0.75 0.00 AX: AZCL-arabinoxylan βG: AZCL-β-glucan Dex: AZCL-dextranHEC: AZCL-HE-cellulose Gal: AZCL-patato galactan GM: AZCL-galactomannanXly: AZCL-xylan XG: AZCL-xyloglucan

Thielavia terrestris CEL7F endoglucanase possessed activity towardAZCL-β-glucan and AZCL-HE-cellulose after 1 hour. Thielavia. terrestrisCEL7F endoglucanase also showed activity on the arabinoxylan, xylan, andxyloglucan dyed substrates, and low activity on the galactomannan dyedsubstrate after a 92 hour incubation.

Example 10 Hydrolysis of Pretreated Corn Stover with the Thielaviaterrestris CEL7F Endoglucanase

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using dilute sulfuric acid. Thefollowing conditions were used for the pretreatment: 0.048 g sulfuricacid/g dry biomass at 190° C. and 25% w/w dry solids for around 1minute. The water-insoluble solids in the pretreated corn stover (PCS)contained 52% cellulose, 3.6% hemicellulose, and 29.8% lignin. Celluloseand hemicellulose were determined by a two-stage sulfuric acidhydrolysis with subsequent analysis of sugars by high performance liquidchromatography using NREL Standard Analytical Procedure #002. Lignin wasdetermined gravimetrically after hydrolyzing the cellulose andhemicellulose fractions with sulfuric acid using NREL StandardAnalytical Procedure #003. Prior to enzymatic hydrolysis, the PCS waswashed with a large volume of double distilled water until the pH washigher than 4.0, and was then sieved through a 100-mash sieve andautoclaved at 121° C. for 30 minutes.

Hydrolysis of PCS was conducted in 96-deep-well plates, (AxygenScientific, Union City, Calif.) sealed by a plate sealer (ALPS-300,Abgene, Epsom, UK), with a total reaction volume of 1.0 ml. Hydrolysisof PCS (10 mg/ml in 50 mM sodium acetate pH 5.0 buffer) was performedusing 1.25 mg of Thielavia terrestris CEL7F endoglucanase (prepared asdescribed in Example 9) per gram of PCS. Broth from Aspergillus oryzaeJal250 (prepared as described in Example 9) was run as a control. PCShydrolysis was performed at 50° C., pH 5.0. Reactions were run induplicates and aliquots were taken during the course of hydrolysis. PCShydrolysis reactions were stopped by mixing a 20 μl aliquot of eachhydrolyzate with 180 μl of 0.11 M NaOH (stop reagent). Appropriateserial dilutions were generated for each sample and the reducing sugarcontent determined using a para-hydroxybenzoic acid hydrazide (PHBAH,Sigma, St. Louis, Mo.) assay adapted to a 96 well microplate format asdescribed below. Briefly, a 90 μl aliquot of an appropriately dilutedsample was placed in a 96 well conical bottomed microplate. Reactionswere initiated by adding 60 μl of 1.5% (w/v) PHBAH in 2% NaOH to eachwell. Plates were heated uncovered at 95° C. for 10 minutes. Plates wereallowed to cool to room temperature (RT) and 50 μl of distilled H₂Oadded to each well. A 100 μl aliquot from each well was transferred to aflat bottomed 96 well plate and the absorbance at A410 nm measured usinga SpectraMax Microplate Reader (Molecular Devices, Sunnyvale, Calif.).Glucose standards (0.1-0.0125 mg/ml diluted with 0.4% sodium hydroxide)were used to prepare a standard curve to translate the obtainedA_(410nm) values into glucose equivalents. The resultant equivalentswere used to calculate the percentage of PCS cellulose conversion foreach reaction. The degree of cellulose conversion to reducing sugar(conversion, %) was calculated using the following equation:Conversion_((%)) =RS _((mg/ml))*100*162/(Cellulose_((mg/ml))*180)=RS_((mg/ml))*100/(Cellulose_((mg/ml))*1.111)In this equation, RS is the concentration of reducing sugar in solutionmeasured in glucose equivalents (mg/ml), and the factor 1.111 reflectsthe weight gain in converting cellulose to glucose.

PCS hydrolysis by the Thielavia terrestris CEL7F endoglucanase (1.25mg/g PCS) yielded a cellulose conversion of 2.1% after 120 hours.Aspergillus oryzae Jal250 (1.25 mg/g PCS) yielded less than 1%conversion after 120 hours.

Example 11 Hydrolysis of Soluble Beta-Glucan from Barley by Thielaviaterrestris Endoglucanase

The Thielavia terrestris Cel7F endoglucanase was tested in the form ofthe Aspergillus oryzae Jal250AILo22 broth described in Example 8. Thebroth was concentrated and exchanged to 50 mM sodium acetate pH 5.0using Centricon Plus-20 centrifugal filter with Biomax-5polyethersulfone membrane (5000 NMWL) from Millipore (Bedford, Mass.).Broth from Aspergillus oryzae Jal250 (vector alone) was treated the sameas above.

The protein concentration in the enzyme solutions was determined usingthe Bicinchoninic Acid (BCA) microplate assay according to themanufacturer's instructions for a BCA Protein Assay Reagent Kit (PierceChemical Co., Rockford, Ill.).

Enzyme dilutions were prepared fresh before each experiment from stockenzyme solutions, which were stored at −20° C.

The activity of the Thielavia terrestris Cel7F endoglucanase on solublebeta-glucan from barley (medium viscosity, 230 kDa, MegazymeInternational Ireland Ltd., Bray, Ireland) was determined at pH 5.5 (50mM sodium acetate with 0.02% sodium azide) and 60° C. The results werecompared with those for Trichoderma reesei Cel7B (EGI) endoglucanase.Recombinant Trichoderma reesei Cel7B (EGI) endoglucanase can be preparedaccording to Takashima et al., 1998, Journal of Biotechnology 65:163-171.

The initial concentration of beta-glucan in the hydrolysis reactions was1.0% (w/v). One ml reactions were run without stirring in Eppendorf 96DeepWell Plates (1.2 ml, VWR Scientific, West Chester, Pa.). The enzymeswere used at three protein loadings, 0.05, 0.1, and 0.2 mg per g ofglucan. In control reactions, the endoglucanases were substituted with50 mM sodium acetate pH 5.5 containing 0.02% sodium azide (buffercontrol) or with concentrated and buffer exchanged Aspergillus oryzaeJal250 broth containing no recombinantly expressed enzymes (Jal250control).

Aliquots were removed from the hydrolysis reactions at 2 hours and 24hours, diluted with deionized water, and analyzed for reducing sugarsusing the p-hydroxybenzoic acid hydrazide (PHBAH) assay as described inExample 10. The relative conversion of beta-glucan as a function ofprotein loading at two incubation times, 2 hours and 24 hours, is shownin FIGS. 7 and 8, respectively. The relative conversion is shown as apercentage of conversion obtained after 24-hour hydrolysis ofbeta-glucan by Thielavia terrestris Cel7F endoglucanase (0.2 mg proteinper g of glucan).

The Thielavia terrestris Cel7F endoglucanase showed higher conversion ofbeta-glucan than the Trichoderma reesei Cel7B endoglucanase andcontinued to produce new reducing end-groups beyond the 2 hourincubation time. In contrast, the Trichoderma reesei Cel7B endoglucanaseshowed almost no additional increase in reducing sugar concentrationafter 2 hours of hydrolysis.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli pTter7F NRRL B-30837Apr. 11, 2005

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolate polypeptide having endoglucanase activity, selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 95% sequence identity with the mature polypeptide of SEQ ID NO:2; (b) a polypeptide encoded by a polynucleotide which hybridizes under high stringency conditions with the full length complementary strand of (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO:1; and (c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:1.
 2. The polypeptide of claim 1, which is encoded by the polynucleotide contained in plasmid pTter7F which is contained in E. coli NRRL B-30837.
 3. The polypeptide of claim 1, wherein the mature polypeptide is amino acids 18 to 336 of SEQ ID NO:
 2. 4. The polypeptide of claim 1, wherein the mature polypeptide coding sequence is nucleotides 52 to 1008 of SEQ ID NO:
 1. 5. The polypeptide of claim 1, further having enzyme activity toward one or more substrates selected from the group consisting of xylan, xyloglucan, arabinoxylan, 1,4-galactan, galactomannan, dextran, and chitin.
 6. A method for producing the polypeptide of claim 1 comprising: (a) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
 7. A method for producing a protein comprising: (a) cultivating a recombinant host cell under conditions conducive for production of the protein, wherein the recombinant host cell comprises a nucleic acid construct comprising a gene encoding a protein operably linked to a nucleotide sequence encoding a signal peptide comprising or consisting of amino acids 1 to 17 of SEQ ID NO: 2, wherein the gene is foreign to the nucleotide sequence; and (b) recovering the protein.
 8. A detergent composition comprising the polypeptide of claim 1 and a surfactant.
 9. The polypeptide of claim 1, which comprises an amino acid sequence having at least 95% sequence identity with the mature polypeptide of SEQ ID NO:
 2. 10. The polypeptide of claim 9, which comprises an amino acid sequence having at least 97% sequence identity with the mature polypeptide of SEQ ID NO:
 2. 11. The polypeptide of claim 1, which is encoded by a polynucleotide which hybridizes under high stringency conditions with the full length complementary strand of (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO:1.
 12. The polypeptide of claim 1, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:
 1. 13. The polypeptide of claim 12, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 97% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:
 1. 